Oxidative metabolism in respiring cells generates highly reactive superoxide (O.sub.2.sup.-) radicals which cause cellular damage (see e.g., Pasquier et al. (1984) Inflammation 8:27-32). A group of metalloenzymes known as superoxide dismutases (SOD) help defend against such cellular damage by catalyzing the oxidation-reduction reaction 2O.sub.2.sup.- +2H.sup.+ .fwdarw.H.sub.2 O.sub.2 +O.sub.2, thus acting as antioxidants.
There are several known forms of SOD, both eukaryotic and prokaryotic. These forms include different metals, such as iron, manganese, copper and zinc. Eukaryotic cells, in particular, contain two forms of SOD. The first form is copper-zinc SOD (Cu/Zn SOD) which is found in the cytosol. The second form is manganese SOD (Mn SOD) which is found in the mitochondria and has also been detected in the cytosol of liver cells (see e.g., McCord et al. "Superoxide and Superoxide Dismutases," Academic Press, N.Y., 1977).
Evidence indicates that SOD may be clinically useful as an antioxidant in many clinical applications, such as protection against ischemia, chemotoxicity (e.g., from anti-cancer agents) and inflammation (see e.g., Oberley et al. (1979) Cancer Res. 39:1141-1149; Huber et al. (1980) Clinics in Rheum Dis. 6:465-498; and McCord et al. (1982) Physiol. Pharma. 60:1346-1352). Specifically, deficiency of human Mn SOD has been linked to the development of clinical rheumatoid arthritis (Pasquier et al., supra.). In addition, human Mn SOD has been suggested to protect against alcohol-induced liver damage (Del Villano et al. (1980) Science 207:991-993), and has been shown in vitro to protect human phagocytosing polymorphonuclear leukocytes from superoxide free radicals more effectively than bovine or porcine Cu/Zn SOD (McCord et al., supra.).
A full-length 196 amino acid sequence for human Mn SOD isolated from human liver cells has been published by Barra et al. (1984) J. Biol. Chem. 259:12595-12601. This sequence differs from the cloned full-length (mature) human Mn SOD amino acid sequences disclosed and claimed in each of U.S. Pat. No. 5,260,204 (Heckl et al.), and U.S. Pat. No. 5,246,847 (Hartman et al.). Specifically, the 196 amino acid sequence of Barra et al. contains a Glu residue instead of a Gln residue at positions 42, 88, 109 and 131. The 196 amino acid sequence of Barra et al. also differs in that it lacks amino acids Gly and Trp between positions 123 and 124 contained in the sequences of U.S. Pat. No. 5,260,204 (Heckl et al.), and U.S. Pat. No. 5,246,847 (Hartman et al.). Moreover, the cloned human Mn SOD amino acid sequence in U.S. Pat. No. 5,260,204 (Heckl et al.) differs from the cloned sequence in U.S. Pat. No. 5,246,847 (Hartman et al.) in that it lacks a Met residue at the N-terminus and contains Glu instead of Gln at residue 146. These differences in amino acid sequence between naturally occurring sequences may be due to the existence of naturally occurring polymorphic variants of human Mn SOD which differ in amino acid sequence due to allelic variation.
The enzymatic activity of natural (wild-type) human and bacterial Mn SOD exhibits a biphasic pattern in the decay of O.sub.2.sup.- under conditions for which the ratio [O.sub.2.sup.- ]/[E] is greater than approximately 10, as observed by stopped-flow (Bull et al. (1991) J. Am. Chem. Soc. 113:4069-4076; McAdam et al. (1977) Biochem. J. 165:71-79) and pulse radiolysis (Hsu et al. (1996) J. Biol. Chem. 271:17687-17691). This biphasic pattern constitutes an initial burst of catalysis followed by a much slower zero-order rate of the disproportionation of superoxide, representing an inhibited phase. Therefore, while initially highly active upon binding its substrate, natural Mn SOD is rapidly inhibited by product hydrogen peroxide (H.sub.2 O.sub.2), causing it to become substantially inactive.
Accordingly, it is an object of the present invention to provide novel Mn SOD proteins which exhibit reduced or no product inhibition compared to their naturally occurring counterparts, and therefore which act as more efficient antioxidants. It is a further object of the invention to provide Mn SOD proteins which exhibit reduced or no product inhibition compared to their naturally occurring counterparts, while maintaining the same or higher catalytic activity compared to their naturally occurring counterparts, and/or which are more stable than their naturally occurring counterparts. It is a further object of the invention to provide nucleic acids encoding Mn SOD proteins of the invention, as well as expression vectors capable of expressing Mn SOD proteins of the invention e.g., following transfection into a host cell (e.g., a bacterial or mammalian cell). It is a further object of the invention to provide methods of using Mn SOD proteins of the invention as therapeutic antioxidative agents to protect cells against damage caused by free oxygen radicals. These and other objects will be apparent from the following summary and description.